Generally, two basic types of metabolites are synthesised in cells, i.e. those referred to as primary metabolites and those referred to as secondary metabolites. A primary metabolite is any intermediate in, or product of the primary metabolism in cells. The primary metabolism in cells is the sum of metabolic activities that are common to most, if not all, living cells and are necessary for basal growth and maintenance of the cells. Primary metabolism thus includes pathways for generally modifying and synthesising certain carbohydrates, proteins, fats and nucleic acids, with the compounds involved in the pathways being designated primary metabolites.
In contrast hereto, secondary metabolites usually do not possess a basal function in cell growth and maintenance. They are a group of chemically very diverse products that often have a restricted taxonomic distribution. Secondary metabolites normally exist as members of closely related chemical families, usually of a molecular weight of less than 1,500, although some bacterial toxins are considerably larger. Two examples of fungal cell secondary metabolites are penicillin and ergotamine.
Plant metabolites include a diverse array of chemically unrelated compounds such as carbohydrates and lipids (e.g. mono-, oligo- and polysaccharides, sugar alcohols, organic acids, fatty acids and lipids, acetylenes and thiophenes), nitrogen-containing compounds (e.g. amino acids, amines, glycosides, glucosinolates, purines, pyrimidines and polypeptides) of which most, but not all, generally are referred to as primary metabolites. Accordingly, some compounds such as fatty acids, sugars and steroids may e.g. be categorised both as primary metabolites and secondary metabolites (see e.g. Dewick, P. M., 1997, Medicinal Natural Products A Biosynthetic Approach, John Wiley & Sons, Chichester).
Secondary plant metabolites include e.g. alkaloid compounds (e.g. terpenoid indole alkaloids and indole alkaloids), phenolic compounds (e.g., quinones, lignans and flavonoids), terpenoid compounds (e.g. monoterpenoids, iridoids, sesquiterpenoids, diterpenoids and triterpenoids). In addition, secondary metabolites include small molecules (i.e., having a molecular weight of less than 600), such as substituted heterocyclic compounds which may be monocyclic or polycyclic, fused or bridged.
Many plant secondary metabolites have value as pharmaceuticals, food colours, flavours and fragrances. Plant pharmaceuticals include e.g.: taxol, digoxin, colchicine, codeine, morphine, quinine, shikonin, ajmalicine and vinblastine. Examples of secondary metabolites that are useful as food additives include anthocyanins, vanillin, and a wide variety of other fruit and vegetable flavours and texture modifying agents.
Plant secondary metabolites such as terpenoid indole alkaloids (TIA) represent a class of pharmaceutically useful compounds which naturally occur in many plant species.
Some plant secondary metabolites are linked to plant or plant cell defence mechanism and may e.g. confer to the plant antimicrobial activity, protection against UV light, herbivores, pathogens, insects and nematodes, and the ability to grow at low light intensity.
There are numerous examples of the application of plant secondary metabolites such as TIA's in medicine. The monomeric alkaloids serpentine and ajmalicine found in Catharanthus roseus are e.g. used as tranquillisers and to reduce hypertension, respectively. The dimeric alkaloids vincristine and vinblastine, also found in C. roseus, are potent antitumor drugs. Camptothecin, a monomeric TIA found in Camptotheca acuminata, also possesses anti-tumor activity. Quinine from Cinchona officinalis is used in malaria treatment.
However, a major problem associated with the industrial use of the above metabolites is that only very small or variable amounts of these compounds are present in plants. The recovery of useful metabolites from their natural sources is thus in many instances difficult due to the enormous amounts of source material which may be required for the isolation of utilisable quantities of the desired products. As an example, over 500 kg of Catharanthus roseus is needed to obtain 1 g of vincristine. Extraction is both costly and tedious, requiring large quantities of raw material and extensive use of chromatographic fractionation procedures. The low levels of these compounds in plants may also imply that many of the compounds are not detected when performing normal screening procedures, hence many unknown compounds may exist.
Biosynthesis of TIA compounds proceeds in most plants via many enzymatic steps. TIA compounds consist of an indole moiety provided by tryptamine and a terpenoid portion provided by the iridoid glucoside compound secologanin. Tryptamine is derived from primary metabolism by a single enzymatic conversion of the amino acid tryptophan, a reaction catalysed by the enzyme tryptophan decarboxylase (TDC) (EC 4.1.1.28). The biosynthesis of secologanin requires a number of enzymatic conversions of which the first step is the hydroxylation of geraniol by the enzyme geraniol 10-hydroxylase (G10H). Tryptamine and secologanin are condensed by the enzyme strictosidine synthase (STR) (EC 4.3.3.2) to form strictosidine, which is the general precursor of all terpenoid indole alkaloids found in plants. The first enzymatic conversion of strictosidine into strictosidine aglucon is catalysed by strictosidine-β-D-glucosidase (SGD) (EC 3.2.1.105). Many different enzyme activities convert strictosidine aglucon into the large variety of terpenoid indole alkaloid end products.
The best progress on molecular characterisation of the terpenoid indole alkaloid pathway has been made with C. roseus or Madagascar periwinkle, a member of the Apocynaceae family. C. roseus cells have the genetic potential to synthesise over a hundred terpenoid indole alkaloids. The biosynthesis of terpenoid indole alkaloids is strongly regulated, and depends on plant cell type and environmental conditions. Their biosynthesis is e.g. induced by fungal elicitors, jasmonates and auxin starvation. Many monomeric TIA compounds are found in all plant organs, but vindoline and vindoline-derived dimeric alkaloids are only found in chloroplast-containing plant tissues.
Until now, only a limited number of genes coding for enzymes involved in terpenoid indole alkaloid biosynthesis have been cloned, such as cDNA clones encoding STR and TDC isolated from C. roseus. In addition, cDNA clones for NADPH:cytochrome P450 reductase (CPR), which is essential for the G10H-catalysed reaction, and SGD have been isolated.
Gene expression studies by the present inventors have shown that the regulation of terpenoid indole alkaloid biosynthesis is controlled largely, if not uniquely, at the level of the expression of biosynthetic genes. Accordingly, analysis of the expression of the terpenoid indole alkaloid biosynthetic genes Tdc and Str1 showed that their expression is low, especially in cell cultures. It was found, that the level of gene expression is likely to be limiting for alkaloid production. Overexpression of a single biosynthetic gene (Tdc or Str1) in transgenic C. roseus cells resulted in significantly elevated levels of the corresponding enzyme activity. However, this did not result in elevated alkaloid levels, presumably because many other enzymes are limiting and would need to be overexpressed.
These studies have further demonstrated what is already known, that the genes are coordinately regulated depending on cell type or environmental conditions. Str1 and Tdc mRNA accumulate in suspension-cultured cells after auxin starvation or phosphate starvation, exposure to fungal elicitors or (methyl) jasmonate and their distribution in the plant is developmentally regulated with the highest levels in the roots. In leaves Tdc and Str1 are induced by a UV-B light pulse. Cpr mRNA accumulation is rapidly induced by fungal elicitor.
The observations that mRNA from genes involved in the biosynthesis of TIA compounds such as Tdc and Str1 mRNAs coordinately accumulate in response to fungal elicitors, (methyl)-jasmonate, UV light, auxin depletion, phosphate depletion, and have similar tissue-specific distributions, have led the present inventors to hypothesise that the Tdc and Str1 genes might be controlled by one or more common regulating factor(s) or substance(s). It was further hypothesised that among possible regulating factors, transcription factors could have such a regulating effect.
It is known that certain transcription factors can regulate complex cell differentiation processes in animals involving numerous target genes. A notable example is muscle differentiation, where either one of a set of myogenic bHLH transcription factors (MyoD, myogenin, Myf5, MRF4) in combination with the MADS-domain transcription factor MEF2 induces muscle cell differentiation and switches on numerous muscle-specific genes. Other examples include homeodomain transcription factors in the fruit fly that regulate cell processes resulting in the determination of segment identity.
The pathway that is most extensively studied in plant secondary metabolism at the transcriptional level, is the one leading to the formation of the anthocyanin pigments. The genes encoding flavonoid biosynthetic enzymes are controlled by a combination of two distinct transcription factor species, one of which has homology to the protein encoded by the vertebrate proto-oncogene c-Myb, and the other with the vertebrate bHLH protein encoded by the proto-oncogene c-Myc. These transcription factors bind to specific sequences in the promoters of the target genes.
The DNA-binding domain of plant MYB proteins consists of two, or for some of them, one imperfect repeat(s). The MYC proteins have a bHLH-type (basic helix-loop-helix) DNA-binding domain and recognise variants of the sequence CANNTG. About ten enzymes are involved in the biosynthesis of anthocyanins starting from phenylalanine. In maize, the entire set of genes encoding these enzymes are thought to be regulated coordinately by the Myc gene R and the Myb gene C1 in the aleurone (epidermal layer of the kernel endosperm), and by homologous genes in other parts of the plant.
Overexpression of the maize Lc gene encoding a MYC-type regulatory protein in Petunia upregulated the whole flavonoid biosynthetic pathway starting from Chs and including the earlier and later genes, resulting among others in intensely pigmented leaves. The expression of the general phenylpropanoid genes Pal and C4h was not affected by Lc overexpression, indicating that Lc only regulates structural genes in the flavonoid branch.
Introduction of the maize R and C1 MYC-type regulators in Arabidopsis intensified pigmentation in normally pigmented tissues and induced pigmentation in plant tissues that are normally unpigmented. In maize cell suspension, ectopic expression of C1 and R led to the accumulation of anthocyanins and a number of other related 3-hydroxy flavonoids. In addition, six anthocyanin structural genes that are targets for Cl/R were expressed at high levels in the transgenic cell line.
In the plant Arabidopsis thaliana, it has been shown that overexpression of the transcription factor CBF-1 that belongs to the AP2 domain class transcription factors resulted in coordinate upregulation of a set of cold-regulated genes. However, there are no suggestions that transcription factors of this class may have an effect on the biosynthesis of metabolites in plant cells and other cells.
The present inventors have now discovered that transcription factors having an AP2 DNA-binding domain are highly useful as central regulators of complex metabolite pathways involving numerous target genes for such transcription factors. This discovery has opened up for providing novel effective means of generating novel metabolite compounds, significantly enhancing the yield of commercially valuable metabolite compounds and also for enhancing the tolerance of plants towards exogenous stress factors and conditions.
Besides such activation of metabolic pathways for the production of metabolites, it is also envisaged that the method of the invention may be used for such purposes as:                study of (plant) metabolism, in which the method of the invention may for instance be used to activate the metabolic pathway(s) under investigation;        study of gene expression, including but not limited to expression profiling, in that the method of the invention allows (the simultaneous) overexpression of one or more genes involved in one or more metabolic pathway(s), the expression or gene products of which may then be studied/determined;        to determine whether one or more specific (usually secondary) metabolites are formed in a plant, by enhancing the levels of said metabolites present in and/or formed by the plant (e.g. from a level below the detection limit of the assay/equipment used to a level above said limit);        lowering or removal of unwanted or toxic metabolites present in a plant or plant material;        discovery enzymes or regulators thereof involved in primary or secundary metabolism.        
Further applications will become clear to the skilled person on the basis of the description given hereinbelow.